Lysine Decarboxylase Broth

Intended Use

Lysine Decarboxylase Broth is used for differentiating microorganisms based on lysine decarboxylation.

Summary and Explanation

Moeller introduced the decarboxylase media for detecting the production of lysine and ornithine decarboxylase and arginine dihydrolase.^1-3 These media are a useful adjunct to other biochemical tests for the speciation and identification of the Enterobacteriaceae and other gram-negative bacilli.^4-8 The production of ornithine decarboxylase is particularly useful for differentiating Klebsiella and Enterobacter species. Klebsiella species are non-motile and, except for K. ornithinolytica, do not produce ornithine decarboxylase, while most Enterobacter species are motile and, except for E. agglomerans, usually produce this enzyme.⁶
Falkow obtained valid and reliable results with a lysine decarboxylase medium he developed to differentiate and identify Salmonella and Shigella.⁹ Although his modification of the Moeller formula was originally described as a lysine medium only, further study by Falkow and then by Ewing, Davis and Edwards,10 substantiated the use of the medium for ornithine and arginine decarboxylase reactions as well.
Ewing, Davis and Edwards¹⁰ compared the Falkow decarboxylase medium base to the Moeller medium and reported that, although the two methods compared favorably in most cases, the Moeller medium was found to be more reliable for cultures of Klebsiella and Enterobacter. They concluded that the Moeller method should be regarded as the standard or reference method, although the Falkow fomula is suitable for determining decarboxylase reactions for most members of the Enterobacteriaceae except for Klebsiella and Enterobacter. The Moeller medium is also particularly useful in the identification of Aeromonas, Plesiomonas, Vibrio spp. and nonfermentative gram-negative bacilli.¹¹
Decarboxylase tests are important in the differentiation and identification of a wide variety of microorganisms and are outlined in numerous standard methods.^12-15
Decarboxylase Base Moeller conforms with the Moeller formulation while Decarboxylase Medium Base is prepared according to the formula described by Falkow. Lysine Decarboxylase Broth is the Falkow medium with L-lysine added in 0.5% concentration.

Principles of the Procedure

Decarboxylase basal media consist of peptones and beef or yeast extract to supply the nitrogenous and other nutrients necessary to support bacterial growth. Pyridoxal is an enzyme co-factor for the amino acid decarboxylase. Dextrose is a fermentable carbohydrate. Bromcresol purple and cresol red are pH indicators. The amino acids lysine, ornithine or arginine are added to the basal medium at a concentration of 10.0 g/L to detect the production of the enzyme specific for these substrates.
When the medium is inoculated with a bacterium that is able to ferment dextrose, acids are produced that lower the pH of the medium and change the color of the indicator from purple to yellow. The acidic condition also stimulates decarboxylase activity. If the organism produces the appropriate enzyme, the amino acid in the medium is degraded, yielding a corresponding amine. Decarboxylation of lysine yields cadaverine, while decarboxylation of ornithine yields putrescine. Arginine is first hydrolyzed to form ornithine, which is then decarboxylated to form putrescine. The production of these amines elevates the pH of the medium, changing the color of the indicator from yellow to purple or violet. If the organism does not produce the appropriate enzyme, the medium remains acidic (yellow). Consult the reference for more information.¹⁶
Each isolate to be tested must also be inoculated into a tube of the basal medium that does not contain the amino acid. If this tube becomes alkaline, the test is invalid.
To obtain the appropriate reactions, the inoculated tubes must be protected from air with a layer of sterile mineral oil. Exposure to air may cause alkalinization at the surface of the medium, which could cause a decarboxylase-negative organism to appear positive.

User Quality Control

Identity Specifications
Lysine Decarboxylase Broth
Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:                        1.4% solution, soluble in purified water upon boiling.
                                     Solution is purple, clear.
Prepared Appearance:     Purple, clear.
Reaction of 1.4%
Solution at 25°C:            pH 6.8 ± 0.2
Cultural Response
Lysine Decarboxylase Broth
Prepare the medium per label directions. Inoculate tubes, overlaying with sterile mineral oil, and incubate at 35 ± 2°C for 18-48 hours. Purple color indicates a positive decarboxylase reaction; a yellow color is negative.

Formula

Lysine Decarboxylase Broth
Approximate Formula* Per Liter
Peptone....................................................................... 5.0 g
Yeast Extract................................................................ 3.0 g
Dextrose...................................................................... 1.0 g
L-Lysine....................................................................... 5.0 g
Bromcresol Purple....................................................... 0.02 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

Lysine Decarboxylase Broth
1. Suspend 14 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Inoculate the broth media by transferring one or two colonies from the surface of a fresh culture with an inoculating loop or needle and mix to distribute the culture throughout the medium. Overlay the medium in each tube with 1 mL sterile mineral oil.
Incubate the tubes with caps tightened at 35 ± 2°C. Examine for growth and decarboxylase reactions after 18-24, 48, 72 and 96 hours before reporting as negative. The medium will become yellow initially, if the dextrose is fermented, and then will gradually turn purple if the decarboxylase or dihydrolase reaction occurs and elevates the pH.

Expected Results

Compare the color of tubes of media containing the specific amino acids with the color of control tubes of basal media (without amino acid) that have been inoculated with the same isolate. If inoculated control tubes show an alkaline reaction, the test is invalid; i.e., either improperly performed or the test organisms can degrade the peptone sufficiently to produce an alkaline reaction in the absence of a specific amino acid.
The medium becomes purple to violet if the reaction is positive (alkaline). A yellow color indicates a negative test; i.e., the organism does not produce the appropriate enzyme.

Limitations of the Procedure

1. If isolated or received on a selective medium, the organism should be subcultured to Trypticase™ Soy Agar with 5% Sheep Blood or other suitable culture medium before attempting to determine decarboxylase or dihydrolase activity.
2. Biochemical characteristics of the Enterobacteriaceae serve to confirm presumptive identification based on cultural, morphological, and/or serological findings. Therefore, biochemical testing should be attempted on pure culture isolates only and subsequent to differential determinations.
3. The decarboxylase reactions are part of a total biochemical profile for members of the Enterobacteriaceae and related organisms. Results obtained from these reactions, therefore, can be considered presumptively indicative of a given genus or species. However, conclusive and final identification of these organisms cannot be made solely on the basis of the decarboxylase reactions.
4. If layers of yellow and purple appear after incubation, shake the test tube gently before attempting to interpret results.
5. If a reaction is difficult to interpret, compare the tube in question to an uninoculated control tube. Any trace of purple after 24 hours of incubation is a positive test.
6. A gray color may indicate reduction of the indicator. Additional indicator may be added before the results are interpreted.¹²
7. Salmonella gallinarum gives a delayed positive ornithine decarboxylase reaction, requiring 5-6 days incubation.³ Many strains of E. coli, including those that ferment adonitol, may exhibit a delayed reaction.³
8. Decarboxylase Medium Base is not satisfactory for the determination of lysine decarboxylase activity with the two genera Klebsiella and Enterobacter.
9. The lysine decarboxylase activity in Salmonella is used to differentiate this group from Citrobacter freundii. Salmonella Paratyphi A, however, gives an atypical negative reaction (yellow color of medium) in 24 hours when Decarboxylase Medium Base is used.⁴

*Store at 2-8° C.

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